Fabp4 as a therapeutic target in skin diseases

ABSTRACT

Provided are methods of regulating proliferation and/or differentiation of keratinocytes and immune cells, more specifically to methods of treating pathologies characterized by hyperproliferative keratinocytes or inflammatory skin diseases by administration of FABP4-inhibitor.

SEQUENCE LISTING

The Sequence Listing submitted in text format (.txt) filed on Jan. 7,2020, named “SequenceListing.txt”, created on Dec. 16, 2019 (2.36 KB),is incorporated herein by reference.

TECHNOLOGICAL FIELD

The present invention relates to methods of regulating proliferationand/or differentiation of keratinocytes and immune cells, morespecifically to methods of treating pathologies characterized byhyperproliferative keratinocytes or inflammatory skin diseases.

BACKGROUND ART

References considered to be relevant as background to the presentlydisclosed subject matter are listed below:

-   [1] Furuhashi et al., Nat Rev Drug Discov 2008, 7(6), 489-503-   [2] Coe et al., Biochim Biophys Acta 1998, 1391(3), 287-306-   [3] Hotamisligil et al., Science 1996, 274(5291), 1377-1379-   [4] Maeda et al., Cell Metab 2005, 1(2), 107-119-   [5] Furuhashi et al., Nature 2007, 447(7147), 959-965-   [6] Tuncman et al., PNAS 2006, 103(18), 6970-6975-   [7] Garin-Shkolnik et al., Diabetes 2014, 63(3), 900-911-   [8] Bolognia et al., Dermatology 2012, Sounders, 3^(rd) ed.-   [9] Krueger et al., Annals of the Rheumatic Diseases 2005(64), 30-36-   [10] Siegenthaler et al., Biochem Biophys Res Commun 1993, 190,    482-487-   [11] Floresta et al., Eur J Med Chem 2017, 138, 854-873-   [12] Cao et al., Cell Metab 2013, 17, 768-778-   [13] Burak et al., Sci Transl Med 2015, 7, 319ra205-   [14] Miao et al., Mol Cell Endocrinol 2015, 403, 1-9-   [15] Won et al., Nat Mater 2014, 13, 1157-1164-   [16] Madsen et al., J Invest Dermatol 1992, 99(3), 299-305-   [17] Guttman-Yassky et al., J Allergy Immunol 2011, 127(5),    1110-1118-   [18] Yuspa et al., J Cell Biol 1989, 109, 1207-1217-   [19] Wu et al., Australasian Journal of Dermatology 2004, 45(1),    47-50-   [20] Van der Fits et al., The Journal of Immunology 2009, 182(9),    5836-5845

Acknowledgement of the above references herein is not to be inferred asmeaning that these are in any way relevant to the patentability of thepresently disclosed subject matter.

BACKGROUND

Fatty acid binding proteins (FABPs) are small cytoplasmic chaperonesthat serve as carriers of hydrophobic molecules, specifically long chainfatty acids and retinoic acid [1,2]. Several members of the FABP familyhave been identified as key regulators of various metabolic functionsand have been linked to the development of insulin resistance, abnormallipid metabolism, and atherosclerosis. FABPs shuttle intracellular fattyacids and other hydrophobic ligands to cellular destinations such asenzymes, membranes, and the nucleus. FABPs are also involved inmodulating intracellular lipid metabolism and regulating geneexpression.

Family members of the intracellular fatty acids chaperones are expressedin a tissue specific manner. FABP4, also termed A-FABP or aP2, is themajor fatty acid binding protein in adipocytes and macrophages. Recentstudies show that FABP4 is central to the pathway that links obesity toinsulin resistance and plays an important role in systemic glucose andlipid metabolism [3,4]. FABP4 has proinflammatory properties inmacrophages. An orally active small-molecule inhibitor of FABP4 wasfound to be an effective therapeutic agent against severeatherosclerosis and type 2 diabetes in mouse models [5]. Additionally,individuals that carry a T87C polymorphism of the FABP4 promoter,leading to a reduced transcriptional activity, have lower serumtriglycerides and a significantly reduced risk of atherosclerosis andtype 2 diabetes as compared with subjects carrying the homozygous WTallele [6].

The transcription factor peroxisome proliferator-activated receptor γ(PPARγ) is a master regulator of genes associated with adipogenesis,insulin responses and immune functions, with favorable outcomesfollowing its activation. PPARγ has been suggested to decrease theinflammatory response of cardiovascular cells, specifically endothelialcells. It has been observed that FABP4 regulates PPARγ expression [7],whereby inhibition of FABP4 increased PPARγ levels in macrophages andadipocytes, and over-expression of FABP4 reduced PPARγ. It was alsoshown that FABP4 reduced PPARγ by triggering its ubiquitination andsubsequent proteasomal degradation. In vivo, FABP4-null mousepre-adipocytes exhibited increased expression of PPARγ and a remarkablyenhanced adipogenesis compared to WT mice, indicating that FABP4regulates the differentiation process. Obesity, mainly visceral obesity,increases morbidity and mortality by promoting insulin resistance,diabetes, and atherosclerosis. It was also found that FABP4 wasincreased and PPARγ was reduced in visceral fat in mice and humans, ascompared to their levels in subcutaneous fat.

The skin functions as a mechanical barrier to the outside world, but italso uses the immune system for protection. Immune responses in theskin, however, are not always protective but can also be harmful innature, and causing diseases [8]. Numerous skin diseases are caused by Tlymphocytes and are therefore immunologically mediated. Consequently,many dermatoses respond favorably to immunosuppressive therapyadministered either systemically or topically. Many of the dermatoseshave a chronic course, and are therefore therapeutically challenging.

Psoriasis is a chronic inflammatory skin disease affecting approximately2-4% of the world population. Its pathogenesis involves both epidermaland immunological functional defects. The hallmarks of psoriasis areabnormal differentiation and hyperproliferation of keratinocytes. Inpsoriasis, the infiltration of inflammatory cells and the activation ofT lymphocytes lead to release of cytokines resulting in proliferation ofkeratinocytes and abnormal differentiation [9].

FABP5, FABP family member also known as epidermal andpsoriasis-associated FABP, was previously found to be increased inpsoriatic skin lesions [10]. However, the role of the metabolicregulator FABP4 in dermatologic diseases in general, and psoriasis inparticular has yet to be observed.

General Description

The inventors of the invention described herein have discovered thatFABP4 is an essential modulator in keratinocytes and immune cells fordevelopment of skin diseases, e.g. inflammatory skin diseases such aspsoriasis. This enables the development of pharmaceutical agents andcompositions that can be useful in inhibiting the development of skindiseases, and may be used to treat such diseases after they havedeveloped.

Thus, in a first aspect of this disclosure, there is provided apharmaceutical composition comprising at least one FABP4-inhibitor foruse in treating or preventing a skin disease or condition in which FABP4is involved.

The FABP4-inhibitor refers to an active agent, which effects FABP4activity, either by decreasing, limiting, or blocking the action,function or expression of the FABP4 protein. Inhibition of FABP4 proteinactivity is to be understood in a broad sense, including range ofinhibitory effects that FABP4-inhibitor may have on the normal (forexample, uninhibited or control) protein activity. Inhibition of proteinactivity may, but need not, result in an increase in the level oractivity of an indicator of the protein's activity (by way of example,this can happen when the protein of interest is acting as an inhibitoror suppressor of a downstream indicator). Thus, FABP4 protein activitymay be inhibited when the level or activity of any direct or indirectindicator of the protein's activity is changed (for example, increasedor decreased) by at least 10%, at least 20%, at least 30%, at least 50%,at least 80%, at least 100%, or at least 250% or more as compared tocontrol measurements of the same indicator. Inhibition of proteinactivity may also be effected, for example, by inhibiting expression ofthe gene encoding the protein or by decreasing the half-life of the mRNAencoding the protein.

According to some embodiments, the FABP4-inhibitor may be selected froma peptide, an antibody, an antibody fragment, a small molecule, a smallinterfering RNA (siRNA), a small hairpin RNA (shRNA), and mixturesthereof.

The term small molecule is meant to denote molecules having a molecularweight of up to about 1000 Da (daltons), at times up to 500 Da.

The small molecule may be a chemical compound, having a ring as its mainstructural feature. The ring may be typically a 3- to 10-membered ring(more typically 5- to 6-membered ring) saturated or unsaturated (i.e.cycloalkyl or aryl), being fully carbonaceous or containing one or morehereoatoms. Namely, the main ring may be a cycloalkyl, a heterocyclyl,an aryl or a heteroaryl, wherein the heteroatom(s) are one or more ofnitrogen, oxygen and sulphur. Alternatively, the small molecule may be asystem of two or more fused rings or having a spiro conjugation, each ofwhich being a cycloalkyl, a heterocyclyl, an aryl or a heteroaryl, eachof the rings having 3- to 10-ring atoms (more typically each having 5-or 6-ring atoms).

In other words, as used herein, cycloalkyl refers to a saturated mono-or multi-cyclic ring system, in certain embodiments of 3 to 10 carbonatoms, in other embodiments of 3 to 6 carbon atoms, in furtherembodiments in other embodiments of 5 or 6 carbon atoms; cycloalkenyland cycloalkynyl refer to mono- or multicyclic ring systems thatrespectively include at least one double bond and at least one triplebond. The ring systems of the cycloalkyl, cycloalkenyl and cycloalkynylgroups may be composed of one ring or two or more rings which may bejoined together in a fused, bridged or sprio-connected fashion.Cycloalk(en)(yn)yl refers to a cycloalkyl group containing at least onedouble bond and at least one triple bond. Heterocyclyl refers to amonocyclic or multicyclic non-aromatic ring system, in one embodiment of3 to 10 members, in another embodiment of 4 to 7 members, in a furtherembodiment of 5 to 6 members, where one or more, in certain embodiments,1 to 4, of the atoms in the ring system is a heteroatom, that is, anelement other than carbon, including but not limited to, nitrogen,oxygen or sulfur. In embodiments where the heteroatom(s) is(are)nitrogen, the nitrogen may be optionally substituted with alkyl,alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl,heterocyclyl, acyl, guanidine, or the nitrogen may be quaternized toform an ammonium group, each being further substituted.

As used herein, aryl refers to aromatic monocyclic or multicyclic groupscontaining from 5 to 10 carbon atoms. Aryl groups include, but are notlimited to groups such as unsubstituted or substituted fluorenyl,unsubstituted or substituted phenyl, and unsubstituted or substitutednaphthyl.

Heteroaryl refers to a monocyclic or multicyclic aromatic ring system,in certain embodiments, of 5 to 18 members where one or more, in oneembodiment 1 to 4, of the atoms in the ring system is a heteroatom, thatis, an element other than carbon, including but not limited to,nitrogen, oxygen or sulfur. The heteroaryl group may be optionally fusedto an aromatic or non-aromatic ring. Heteroaryl groups include, but arenot limited to, furyl, imidazolyl, pyrimidinyl, tetrazolyl, thienyl,pyridyl, pyrrolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl,triazolyl, quinolinyl and isoquinolinyl.

The main ring structure (either containing a single ring or being afused multicyclic system), may be substituted on each of its possiblesubstitution positions by a variety of substituents, e.g. an alkoxy oralkylthio group (RO- or RS- group), a halide (or halogen atom, namely I,Br, Cl and F), a pseudohalide (such as cyanide, cyanate, thiocyanate,selenocyanate, trifluoromethoxy, azide), a haloalkyl, a haloalkoxy, anester (—COOR group), an ether (—R′OR group), an alkanoic acid (—ROOH),amino, sulphinyl (—S(O)-), sulphonyl (—S(O)2-), sulpho (—S(O)20-), mono-or dialkylaminocarbonyl (—C(O)NHR or —C(O)NRR'), carboxamide (—NR′COR),amido (—C(O)NH-), thioamido (—C(S)NH-), oxyamido (—OC(O)NH-), thiaamido(—SC(O)NH-), dithiamido (—SC(S)NH-), ureido (—HNC(O)NH-), thioureido(—HNC(S)NH-), formamido (—NH—C(O)—H), and others.

wherein each of R and R′ a C₁-C₈ straight or branched alkyl, alkylene,aryl or heteroaryl group.

Amino as used herein is meant to encompass primary, secondary ortertiary amines where the point of attachment is through the nitrogenatom which is substituted with C₁-C₆ straight or branched alkyl. In caseof a tertiary amine, the substituent is the same or different.

In some embodiments, the small molecule may be selected from carbazolealkanoic acid or carbazole alkanoic acid derivatives, aryl sulfonamideor aryl sulfonamide derivatives, sulfonylthiophene or sulfonylthiophenederivatives, hydroxypyrimidine, carbazole or carbazole derivatives,indole or indole derivatives, carbazole or carbazole derivatives,benzoylbenzene or benzoylbenzene derivatives, biphenyl-alkanoic acid orbiphenyl-alkanoic acid derivatives, oxazole-alkanoic acid oroxazole-alkanoic acid derivatives, pyrimidine or pyrimidine derivatives,pyrimidone or pyrimidone derivatives, pyridine or pyridine derivatives,pyrazine or pyrazine derivatives, pyrazinone or pyrazinone derivatives,tetrazole and tetrazole derivatives, triazolopyrimidine ortriazolopyrimidine derivatives, triazolopyrimidinone ortriazolopyrimidinone derivatives, pyrazole or pyrazole derivatives,(2-(2′-(5-ethyl-3,4-diphenyl-1H-pyrazol-1-yl)(1,1′-biphenyl)-3-yl)oxy)-aceticacid (BMS309403) and4-{[2-methoxycarbonayl)-5-(2-thienyl)-3-thienyl]amino}-4-oxo-2-butanoicacid (BMS480404), and salts, stereomers, hydrates and mixtures thereof[11].

The term derivative refers to a chemically modified compound derivedfrom a parent compound described herein, that differs from the parentcompound by one or more elements, substituents and/or functional groupssuch that the derivative has the same or similar biologicalproperties/activities as the parent compound, as defined herein. Forexample, a pyrazole derivative may be(2-(2′-(5-ethyl-3,4-diphenyl-1H-pyrazol-1-yl)(1,1′-biphenyl)-3-yl)oxy)-aceticacid (BMS309403).

The term alkanoic acid refers to acids of saturated or unsaturatedcarbon chains, containing from 1 (or 2) to 18 carbons, and are straightor branched, e.g. carboxylic, ethanoic, propanoic, butanoic, pentanoic,hexanoic, heptanoic, etc.

In other embodiments, the small molecule may be selected from carbazolebutanoic acid, aryl sulfonamide, sulfonylthiophene or sulfonylthiophenederivatives, 4-hydroxypyrimidine, 2-hydroxypyrimidine, carbazole orcarbazole derivatives, tetrahydrocarbazole or tetrahydrocarbazolederivatives, 2,3-dimethylindole or 2,3-dimethylindole derivatives,benzoylbenzene, biphenyl-alkanoic acid or biphenyl-alkanoic acidderivatives, 2-oxazole-alkanoic acid or 2-oxazole-alkanoic acidderivatives, tetrahydropyrimidine or tetrahydropyrimidine derivatives,pyridine or pyridine derivatives, pyrazine or pyrazinone derivatives,quinolone or quinolone derivatives, aryl carboxylic acid or arylcarboxylic acid derivatives, tetrazole, triazolopyrimidine ortriazolopyrimidine derivatives, indole or indole derivatives, flavonoids(such as flavanoles, falvanones, isoflavone, pyrazole or pyrazolederivatives,(2-(2′-(5-ethyl-3,4-diphenyl-1H-pyrazol-1-yl)(1,1′-biphenyl)-3-yl)oxy)-aceticacid (BMS309403) and4-{[2-methoxycarbonayl)-5-(2-thienyl)-3-thienyl]amino}-4-oxo-2-butanoicacid (BMS480404), and salts, stereomers, hydrates and mixtures thereof.

Exemplary small molecules which are FABP4 inhibitors are shown in Table1.

TABLE 1 FABP4 inhibitors 1

R¹, R² = H or halogen R³ = H, C₁₋₄-alkyl 2

3

4

R¹ = H or halogen R², R³ = H C₁₋₄-alkyl Ar = phenyl, 2-thienyl 5

R = H, C₁₋₆-alkyl, C₁₋₆-haloalkyl, C₁₋₄-carboxylic acid X = O, NH 6

n = 0-4 7

8

9

X = halogen, C₁₋₄alkyl, C₁₋₄alkoxy 10

X = halogen, C₁₋₄alkyl, C₁₋₄alkoxy 11

n = 0-2 12

13

R = C₁₋₆-alkyl, C₁₋₄-haloalkyl, C₁₋₄-alkyl, CF₃, CONH₂, halogen n = 1-214

X = O, NOH 15

n = 1-2 16

17

18

19

20

21

R¹ = H, C₁₋₄alkyl R² = H, C₁₋₄alkyl, C₁₋₄alkoxy, halogen, NO₂, NH₂ R³ =H, C₁₋₄alkyl, C₁₋₄alkoxy, halogen, NO₂ 22

23

24

25

26

27

R = C₁₋₄alkyl, phenyl, fluorophenyl 28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

In some embodiments, the small molecule may be selected from thosedetailed in table 1.

In some embodiments, the small molecule may be at least one from thosedetailed in table 1.

In some other embodiments, the small molecule is selected from pyrazoleor pyrazole derivatives, (2-(2′-(5-ethyl-3,4-diphenyl-1H-pyrazol-1-yl)(1,1′-biphenyl)-3 -yl)oxy)-acetic acid(BMS309403) and4-{[2-methoxycarbonayl)-5-(2-thienyl)-3-thienyl]amino}-4-oxo-2-butanoicacid (BMS480404), and salts, stereomers, hydrates and mixtures thereof.

In further embodiments, the small molecule may be(2-(2′-(5-ethyl-3,4-diphenyl-1H-pyrazol-1-yl)(1,1′-biphenyl)-3-yl)oxy)-aceticacid (BMS309403). In some other embodiments, the small molecule may be4-{[2-methoxycarbonayl)-5-(2-thienyl)-3-thienyl]amino}-4-oxo-2-butanoicacid (BMS480404).

The FABP4-inhibitor, by some embodiments, may be an antibody thatspecifically binds to and inhibits the activity of FABP4. The antibodyis a polypeptide ligand comprising at least a light chain or a heavychain immunoglobulin variable region, which specifically recognizes andbinds an epitope of an antigen, namely to a FABP4 protein or a fragmentthereof. The term means to denote intact immunoglobulins and theirvariants and portions known in the art, such as Fab′ fragments, F(ab)′2fragments, single chain Fv proteins (“scFv”), and disulfide stabilizedFv proteins (“dsFv”). The term also includes recombinant forms such aschimeric antibodies (for example, humanized murine antibodies), andheretoconjugate antibodies (such as, bispecific antibodies). See also,Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford,Ill.); Kuby, Immunology, 3rd Ed., W.H. Freeman & Co., New York, 1997.

The antibody may be a monoclonal antibody or a polyclonal antibody.Monoclonal antibodies are produced by a single clone of B-lymphocytes orby a cell into which the light and heavy chain genes of a singleantibody have been transfected, including humanized monoclonalantibodies. Polyclonal antibodies are antibodies that are secreted bydifferent B cell lineages within the body.

The antibodies are said to bind specifically to FABP4 protein orfragments thereof; namely, the antibodies are capable of associating ina selective and preferable manner with FABP4 proteins or theirfragments, over binding with other molecular/macromolecular entitieswith which FABP4 proteins and their fragments are admixed. An antibodyhaving inhibitory effect (or neutralizing effect) on FABP4 is anantibody that inhibits or suppresses at least one activity of FABP4 orat least one activity associated with FABP4; for example, by blockingthe binding of FAPB4 to a ligand to which it normally binds, bydisrupting or otherwise interfering with a protein-protein interactionof the FABP4 with another protein, etc.

In some embodiments, the antibody is an anti-FABP4 antibody. Exemplaryanti-FABP4 antibodies are described in the art, and may include, interalia, Ab anti FABP4 [12], Ab CA33 [13], Ab 2E4 [14], and others.

The FABP4-inhibitor may, by some embodiments, be an antisense or a senseinhibitor. Antisense and sense inhibitors are bio-macromolecules thatbind (or hybridize) with messenger RNA (mRNA) produced by specificgenes, e.g. FABP4 encoding gene, and inactivate or modulate theirexpression. The antisense inhibitor is typically an oligomeric compoundthat is at least partially complementary to the region of a specifictarget nucleic acid molecule to which it hybridizes, thus causing RNAinterference (RNAi). Thus, RNAi works through the targeting of mRNA viasequence-specific matches and results in degradation of target mRNA orits translational inhibition, leading to altering or loss of proteinexpression, for example suppression of expression of FABP4. Non-limitingexamples of antisense compounds include primers, probes, antisenseoligonucleotides, siRNAs, miRNAs, shRNA and ribozymes. Either anantisense or a sense inhibitor can be used to target a portion of dsDNA,as both will interfere with the expression of that portion of the dsDNA.The antisense molecule can bind to the plus strand, and the sensemolecule can bind to the minus strand. These compounds can be introducedas single-stranded, double-stranded, circular, branches or hairpincompounds and can contain structural elements such as internal orterminal bulges or loops. Double-stranded antisense compounds can be twostrands hybridized to form double-stranded compounds or a single strandwith sufficient self-complementarity to allow for hybridization andformation of a fully or partially double-stranded compound.

The FABP4-inhibitor may be small interfering RNA (siRNA), which aresmall 19-25 bp dsRNA molecules, with phosphorylated 5′ ends andhydroxylated 3′ ends, with two overhanging nucleotides. An example ofspecific interfering RNA delivery into adipose tissue against celltype—specific carrier molecules is described in [15] (incorporatedherein by reference). In some embodiments, the FABP4-inhibitor is ansiRNA comprising the nucleic acid sequences SEQ ID NO: 2-9:

sense (SEQ ID NO: 2) 3′ guagguaccuggaaacuuguu antisense (SEQ ID NO: 3)5′ caaguuuccagguaccuacuu sense (SEQ ID NO: 4) 3′ gaaaugggauggaaaaucauuantisense (SEQ ID NO: 5) 5′ ugauuuuccaucccauuucuu sense (SEQ ID NO: 6)3′ gaugugaucaccauuaaauuu antisense (SEQ ID NO: 7)5′ auuuaauggugaucacaucuu sense (SEQ ID NO: 8) 3′ gaaagucaagagcaccauauuantisense (SEQ ID NO: 9) 5′ uauggugcucuugacuuucuu.

It is of note that selecting suitable FABP4-inhibitors may be carriedout by any suitable method known per-se (see, for example Hughes et al.,Br J Pharmacol. 2011, 162(6), 1239-1249). For example, active compoundsmay be selected from libraries and screened, based on their selectivityto FABP4 and their resulting potency or activity towards FABP4inhibition.

As further noted above, it has been previously observed by the inventorsof the present invention that PPARγ expression in macrophages andadipocytes is negatively regulated by FABP4, and that FABP4 wasincreased and PPARγ was reduced in inflammatory sites such as thevisceral fat [7]. The inventors have now found that FABP4 isoverexpressed in skin, in T lymphocytes and keratinocytes, and thatthere is a negative correlation between FABP4 and PPARγ expression thereas well, for example in inflammation-involved skin. Therefore, withoutwishing to be bound by theory, the inhibition of FABP4 concomitantlywith activation of PPARγ may have a synergistic effect in treatingvarious skin diseases.

Thus, the pharmaceutical compositions of this disclosure may comprise,or used concomitantly with, at least one PPARγ agonist. The PPARγagonist is an active agent which binds to the peroxisomeproliferator-activated receptor, and activates the receptor.

According to some embodiments, the pharmaceutical composition maycomprise at least one PPARγ agonist. In some embodiments, the at leastone PPARγ agonist is a thiazolidinedione derivative. In otherembodiments, the PPARγ agonist may be selected from Pioglitazone(Actos), Rosiglitazone (Avandia), Lobeglitazone (Duvie), Ciglitazone,Darglitazone, Englitazone, Netoglitazone, Rivoglitazone, Troglitazone(Rezulin), Rhodanine, and other thiazolidinedione derivatives. As a manof the art would appreciate, other PPARγ agonists are also encompassedby the scope of the present disclosure, such as indole-derivatives orvarious non-steroidal anti-inflammatory drugs (e.g. ibuprofen), as wellas other PPARγ agonists.

The pharmaceutical compositions of this disclosure may comprise apharmaceutically acceptable carrier. The pharmaceutically acceptablecarrier may be for example, vehicles, adjuvants, excipients, ordiluents, and are well-known to those who are skilled in the art and arereadily available to the public. See, for example, Remington'sPharmaceutical Science, by E. W. Martin, Mack Publishing Co., Easton,Pa., 15th Edition (1975). It is preferred that the pharmaceuticallyacceptable carrier be one which is chemically inert to the activecompounds and one which has no detrimental side effects or toxicityunder the conditions of use. The choice of carrier will be determined inpart by the particular active agent, as well as by the particular methodused to administer the composition. Accordingly, there is a wide varietyof suitable formulations of the pharmaceutical composition encompassedby the present disclosure.

The pharmaceutically acceptable carriers may be suitable for topical,oral, rectal, vaginal, transdermal, subcutaneous, intravenous,intramuscular, ocular, and intranasal administration of theFABP4-inhibitor. The pharmaceutical compositions are prepared in amanner well known in the pharmaceutical art. In making thepharmaceutical compositions of this disclosure, the components areusually mixed with an excipient, diluted by an excipient or enclosedwithin such a carrier which can be manipulated to the desired form.Based on the particular mode of administration, the pharmaceuticalcomposition may be formulated into tablets, pills, capsules, sachets,granules, powders, chewing gum, suspensions, emulsions, solutions, gels,lotions, oils, soaps, sprays, creams, ointments, films, microcapsules,microspheres, liposomes, vesicles, microemulsions, lipospheres, patches,and ethosomes.

The pharmaceutical composition may, by some embodiments, be adapted fordelivery of the at least one FABP4-inhibitor topically, orally, byinhalation, nasally, transdermally, ocularly or parenterally into thecirculatory system of a subject.

According to some embodiments, the pharmaceutical compositions areadapted for administration of said at least one FABP4-inhibitor byinjection.

According to other embodiments, the pharmaceutical compositions areadapted for oral administration of said at least one FABP4-inhibitor.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the compound, or compositioncomprising same, dissolved in diluents, such as water, saline, or juice(e.g. orange juice); (b) capsules, sachets, tablets, lozenges, andtroches, each containing a predetermined amount of the activeingredient, as solids or granules; (c) powders; (d) suspensions in anappropriate liquid; and (e) suitable emulsions. Liquid formulations mayinclude diluents, such as water and alcohols, for example, ethanol,benzyl alcohol, and the polyethylene alcohols, either with or withoutthe addition of a pharmaceutically acceptable surfactant, suspendingagent, or emulsifying agent. Capsule forms can be of the ordinary hard-or soft-shelled gelatin type containing, for example, surfactants,lubricants, and inert fillers, such as lactose, sucrose, calciumphosphate, and corn starch. Tablet forms can include one or more oflactose, sucrose, mannitol, corn starch, potato starch, alginic acid,microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicondioxide, talc, magnesium stearate, calcium stearate, zinc stearate,stearic acid, and other excipients, colorants, diluents, bufferingagents, disintegrating agents, preservatives, flavoring agents, andpharmacologically compatible carriers. Lozenge forms can comprise theactive ingredient in a flavor, usually sucrose and acacia or tragacanth,as well as pastilles comprising the active formulation in an inert base,such as gelatin and glycerin, or sucrose and acacia, emulsions, gels,and the like containing, in addition to the active formulation, suchcarriers as are known in the art.

The pharmaceutical compositions of this disclosure, being for use intreating or preventing an inflammatory skin disease, may be adapted toinduce a systemic or a non-systemic effect; namely, the compositions maybe adapted to deliver the active agent, FABP4-inhibitor, into thecirculatory system of a subject, or to deliver the active agent to atarget site (e.g. a specific layer of the skin).

As known, human skin is made of numerous layers which may be dividedinto three main group layers: Stratum corneum which is located on theouter surface of the skin, the epidermis, and the dermis. While theStratum comeum is a keratin-filled layer of cells in an extracellularlipid-rich matrix, which in fact is the main barrier to drug deliveryinto skin, the epidermis and the dermis layers are viable tissues. Theepidermis is free from blood vessels, but the dermis contains capillaryloops that can channel therapeutics for transepithelial systemicdistribution.

In some embodiments, the pharmaceutical composition is adapted fortransdermal administration of said at least one FABP4-inhibitor. Inother embodiments, the pharmaceutical compositions are adapted fortopical administration of said at least one FABP4-inhibitor across skinlayers. In some other embodiments, the pharmaceutical compositions areadapted for topical delivery of said at least one FABP-inhibitor acrossthe Stratum comeum.

The pharmaceutical compositions may be formulated in any form suitablefor dermal or topical administration, such as a gel, a lotion, oil,soap, a spray, an emulsion, a cream, an ointment, a solution, asuspension, a film, microcapsules, microspheres, liposomes, vesicles,microemulsions, lipospheres, and patches.

The pharmaceutical compositions of this disclosure are used for treatingor preventing a skin disease or condition in which FABP4 isoverexpressed. In some embodiments, the skin disease or condition may beselected from the group consisting of psoriasis, dermatitis (atopic,seborrheic, contact), eczema, parapsoriasis, lichen planus, lichenplano-pilaris, pityriasis lichenoides et varioliformis acuta, pityriasislichenoides chronica, pityriasis rubra pilaris, pityriasis rosea,graft-versus-host disease, histiocytoses, drug-induced eruptions,autoimmune connective tissue diseases (e.g. lupus), rosacea,folliculitis, acne, warts, ichthyosis, vitiligo, scarring alopecia,cutaneous T cell lymphoma (CTCL), actinic keratosis, squamous cellcarcinoma, basal cell carcinoma, nevus, lichen simplex chronicus,xerosis, keratosis, keratoderma, pruritus, a bum, a scar, a callus, anda keloid.

According to some embodiments, the skin disease is lymphoma (CTCL).

In other embodiments, the pharmaceutical compositions of this disclosureare used for treating or preventing an inflammatory skin disease orcondition. Namely, the compositions cause at least one therapeuticeffect on skin diseases or conditions that involve an inflammatorycomponent in which FABP4 is overexpressed.

In some embodiments, the inflammatory skin disease or condition may beselected from the group consisting of psoriasis, dermatitis (atopic,seborrheic, contact), eczema, parapsoriasis, lichen planus, lichenplano-pilaris, pityriasis lichenoides et varioliformis acuta, pityriasislichenoides chronica, pityriasis rubra pilaris, pityriasis rosea,graft-versus-host disease, histiocytoses, drug-induced eruptions,autoimmune connective tissue diseases (e.g. lupus), rosacea,folliculitis, acne, warts, ichthyosis, vitiligo, scarring alopecia, andCTCL.

According to such embodiments, the inflammatory skin disease ispsoriasis. According to other embodiments, the inflammatory skin diseaseis dermatitis (atopic, seborrheic, contact).

According to another aspect, there is provided a topical formulation fortransdermal delivery of at least one FABP4-inhibitor, said compositioncomprising at least one FABP4-inhibitor and at least onepharmaceutically acceptable carrier. In some embodiments, the topicalformulation may further comprise at least one PPARγ agonist.

A further aspect of the present disclosure provides a method fortreating or preventing a skin disease in a subject (e.g. an inflammatoryskin condition), comprising administering to a subject in need thereof,a therapeutically effective amount of at least one FABP4-inhibitor or apharmaceutical composition comprising thereof. The FABP4-inhibitors foruse in the method of treatment may be selected from those detailedherein.

In a further aspect, there is provided a method for treating orpreventing psoriasis in a subject, comprising administering to a subjectin need thereof, a therapeutically effective amount of at least oneFABP4-inhibitor or a pharmaceutical composition comprising thereof.

Another aspect provides a method for treating or preventing CTCL in asubject, comprising administering to a subject in need thereof, atherapeutically effective amount of at least one FABP4-inhibitor or apharmaceutical composition comprising thereof.

The pharmaceutical compositions of this disclosure may be selected totreat, prevent or diagnose any pathology or condition. The termtreatment or any lingual variation thereof, as used herein, refers tothe administering of a therapeutic amount of the FABP4-inhbitor ofcomposition comprising thereof, which is effective to ameliorateundesired symptoms associated with a disease, to prevent themanifestation of such symptoms before they occur, to slow down theprogression of the disease, slow down the deterioration of symptoms, toenhance the onset of remission period, slow down the irreversible damagecaused in the progressive chronic stage of the disease, to delay theonset of said progressive stage, to lessen the severity or cure thedisease, to improve survival rate or more rapid recovery, or to preventthe disease from occurring or a combination of two or more of the above.

As known, the effective amount for purposes herein may be determined bysuch considerations as known in the art. The amount must be effective toachieve the desired therapeutic effect, depending, inter alia, on thetype and severity of the disease to be treated and the treatmentregimen. The effective amount is typically determined in appropriatelydesigned clinical trials (dose range studies) and the person versed inthe art will know how to properly conduct such trials in order todetermine the effective amount. As generally known, the effective amountdepends on a variety of factors including the affinity of the ligand tothe receptor, its distribution profile within the body, a variety ofpharmacological parameters such as half-life in the body, on undesiredside effects, if any, on factors such as age and gender, and others.

The method of treatment, according to some embodiments, may furthercomprise administering a PPARγ agonist to the subject. PPARγ agonist maybe administered concomitantly with said at least one FABP4-inhibitor, orsequentially to the FABP4-inhibitor.

As used herein, concomitantly (or simultaneously) or any lingualvariation thereof is used to mean that the components of a compositionare administered concurrently, e.g., one together with the other.Simultaneous administration may permit one component in the combinationto be administered within a certain time period (e.g., 5 minutes, 10minutes or even a few hours) after the other, provided that thecirculatory half-life concentration of the first administered componentin a combination is concurrently present in therapeutically effectiveamounts with the other components administered thereafter. The timedelay between administration of the components may vary depending on theexact nature of the components and the formulation containing them, theinteraction between the individual components, their respectivehalf-lives, and on such other factors as easily recognized by the versedartesian.

Sequentially (or separately) or any lingual variation thereof is usedherein to mean that the time period between administering one componentand the other is significant i.e., the first administered component mayno longer be present (or is present in subclinical amounts) in thebloodstream in a therapeutically effective amount when the second (orsubsequent) component is administered.

As noted above, it was discovered by the inventors that FABP4 isoverexpressed in keratinocytes or inflammatory cells (such asmacrophages, and lymphocytes) in a skin tissue biopsy from patients withinflammatory skin disease, but is not significantly expressed innon-involved skin tissue. Accordingly, identification of FABP4overexpression can be utilized to detect early onset inflammatory skindisorders or detect predisposition of a subject to develop aninflammatory skin disease.

Thus, another aspect of the present disclosure provides a method fordetecting the predisposition in a subject for developing an inflammatoryskin disease or condition (or detect early onset of said inflammatoryskin disease or condition), the method comprises detecting expression ofFABP4 in a sample containing keratinocytes or inflammatory cells fromthe subject, wherein the presence of FABP4 in the sample above apre-determined base-level indicates a predisposition for developing theinflammatory skin disease or condition. The inflammatory skin disease orcondition may be any of the diseases and conditions described herein.

In some embodiments, said pre-determined base-level is a level of FABP4in a normal skin sample

The term predisposition refers herein to an effect of a factor orfactors that render a subject susceptible to a condition, disease, ordisorder, such as an inflammatory skin disease. In some embodiments, thedisclosed methods may be used to identify a subject predisposed todeveloping a condition, disease, or disorder.

It is to be understood that detection may be qualitative orquantitative, and may be carried out by any suitable method knownper-se; non-limiting examples of such methods may beimmunohistochemistry, PCR techniques (including RT-PCT and qRT-PCR),Western analysis, in-situ hybridization, and others.

The biological sample may be obtained directly or indirectly from thesubject, including whole blood, plasma, serum, tears, mucus, saliva,urine, pleural fluid, tissues, cells (such as fibroblasts, peripheralblood mononuclear cells, or skin cells), organs, and/or extracts oftissues.

In some embodiments, the biological sample is a skin sample.

It is to be understood that detection of FABP4 in the sample may referto detecting the expression of a FABP4 nucleic acid, expression of aFABP4 protein fragment, and/or expression of a FABP4 protein.

The control level or base-level refers to a reference standard, such asa known value indicative of base concentration or expression of FABP4 ina healthy subject or from a non-involved sample of the subject. Inparticular examples a control sample is taken from a subject that isknown not to have a disease or condition, such as psoriasis. In otherexamples a control is taken from the subject being diagnosed, but at anearlier time point, either before disease onset or prior to or at anearlier time point in disease treatment.

The difference between a test sample and a control can be an increase orconversely a decrease of expression of FABP4 protein, fragment ornucleic acid. The difference can be a qualitative difference or aquantitative difference, for example a statistically significantdifference. In some examples, a different is an increase or decrease,relative to a control, of at least about 10%, such as at least about20%, at least about 30%, at least about 40%, at least about 50%, atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 100%, at least about 150%, at least about 200%, atleast about 250%, at least about 300%, at least about 350%, at leastabout 400%, at least about 450%, or greater than 500%.

In yet another aspect, the present disclosure provides a method fortreating or preventing an inflammatory skin disease in a subject,comprising:

detecting expression of FABP4 in a sample containing keratinocytes orinflammatory cells from the subject,

determining whether FABP4 expression is above or below a pre-determinedbase-level, and

administering to the subject a therapeutically effective amount of atleast one FABP4-inhibitor or a pharmaceutical composition comprisingthereof if the FABP4 expression in the sample is above saidpre-determined base-level.

The subject to be treated or diagnosed refers to both human andnon-human mammals (i.e. both human and veterinary subjects, for example,humans, non-human primates, dogs, cats, horses, and cows).

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise.

As used herein, the term about is meant to encompass deviation of ±10%from the specifically mentioned value of a parameter, such asconcentration, molecular weight, etc.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

BRIEF DESCRIPTION OF THE DESCRIBED SEQUENCES

The nucleic acid sequences provided herewith (Table 2 below) are shownusing standard letter abbreviations for nucleotide bases as defined in37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown,but the complementary strand is understood as included by any referenceto the displayed strand.

TABLE 2 Sequence listing SEQ ID NO: 1 atagcaccct cctgtgctgc (cDNAagcctttctc acctggaaga sequence of cagctcctcc tcgaaggttt mouse FABP4acaaaatgtg tgatgccttt that was gtgggaacct ggaagcttgt inserted intoctccagtgaa aacttcgatg a lentiviral attacatgaa agaagtggga vector)gtgggctttg ccacaaggaa agtggcaggc atggccaagc ccaacatgat catcagcgtaaatggggatt tggtcaccat ccggtcagag agtactttta aaaacaccga gatttccttcaaactgggcg tggaattcga tgaaatcacc gcagacgaca ggaaggtgaa gagcatcataaccctagatg gcggggccct ggtgcaggtg cagaagtggg atggaaagtc gaccacaataaagagaaaac gagatggtga caagctggtg gtggaatgtg ttatgaaagg cgtgacttccacaagagttt atgaaagggc atgagccaaa ggaagaggcc tggatggaaa tttgcatcaaacactacaat agtcagtcgg atttattgtt tttttttaaa gatatgattt tccactaataagcaagcaat taattttttc tgaagatgca ttttattgga tatggttatg ttgattaaataaaacctttt tagacttaga aaaaaa 2 sense 3′-N  guagguaccuggaaacuuguu 3anti-sense 5′-P  caaguuuccagguaccuacuu 4 sense 3′-N gaaaugggauggaaaaucauu 5 anti-sense 5′-P  ugauuuuccaucccauuucuu 6sense 3′-N  gaugugaucaccauuaaauuu 7 anti-sense 5′-P auuuaauggugaucacaucuu 8 sense 3′-N  gaaagucaagagcaccauauu 9anti-sense 5′-P  uauggugcucuugacuuucuu

BRIEF DESCRIPTION OF THE DRAWINGS

In order to better understand the subject matter that is disclosedherein and to exemplify how it may be carried out in practice,embodiments will now be described, by way of non-limiting example only,with reference to the accompanying drawings, in which:

FIGS. 1A-1F show expression of FABP4 and PPARy proteins inimmunohistochemistry staining of human skin: healthy controls skin(FIGS. 1A-1B), psoriasis-involved skin (FIGS. 1C-1D), anddermatitis-involved skin (FIGS. 1E-1F). Tissue sections were stainedwith anti FABP4 (FIGS. 1A, 1C, 1E) or anti PPARγ (FIGS. 1B, 1D, 1F)antibodies. Original magnification ×100.

FIGS. 2A-2C show expression of FABP4 protein in immunohistochemistrystaining of human skin: normal human skin (FIG. 2A), epidermis ofpsoriasis-involved skin (FIG. 2B), and dermis of psoriasis-involved skin(FIG. 2C). Tissue sections were stained with anti-FABP4 antibody.Original magnification ×200.

FIGS. 3A-3B show expression of PPARy protein in immunohistochemistrystaining of human skin: normal human skin (FIG. 3A), andpsoriasis-involved skin (FIG. 3B). Tissue sections were stained withanti-PPARγ antibody. Original magnification ×200.

FIGS. 4A-4B are immunohistochemistry staining of FABP4 protein in humanskin: normal skin (FIG. 4A), and human cutaneous T cell lymphoma (CTCL)skin lesions (FIG. 4B). Tissue sections were stained with anti-FABP4antibody. Original magnification ×200.

FIG. 5 is an immunoblot (Western Blotting) showing levels ofkeratinocyte differentiation markers K1 and K5 in primary murinekeratinocytes overexpressing FABP4. The keratinocytes were infected witha lentiviral vector expressing FABP4 tagged with GFP (FABP4-GFP),lentiviral vector expressing GFP (GFP), or left untreated (noinfection).

FIG. 6 shows Psoriasis Area Severity Index (PASI) scoring of the animalsfrom the in vivo experiment at various time points. *p<0.05; ***p<0.001compared to Vehicle group (using Two-way ANOVA followed by Bonferronipost-hoc test).

FIG. 7 shows erythema severity scoring of the animals from the in vivoexperiment at various time points. ***p<0.001 compared to Vehicle group(using Two-way ANOVA followed by Bonferroni post-hoc test).

FIG. 8 shows skin thickness severity scoring of the animals from the invivo experiment at various time points. **p<0.01; ***p<0.001 compared toVehicle group (using Two-way ANOVA followed by Bonferroni post-hoctest).

FIG. 9 shows scaling severity scoring of the animals from the in vivoexperiment at various time points. *p<0.05, **p<0.01; ***p<0.001compared to Vehicle group (using Two-way ANOVA followed by Bonferronipost-hoc test).

FIGS. 10A-10F are representative pictures of mice on Day 7 of the invivo experiment: Naïve (FIG. 10A), Vehicle (FIG. 10B), BMS 5 mg/kg (FIG.10C), BMS 15 mg/kg (FIG. 10D), BMS 30 mg/kg (FIG. 10E), and Cortisoneacetate (FIG. 10F).

DETAILED DESCRIPTION OF EMBODIMENTS

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the disclosure to the particular features or embodimentsdescribed.

EXAMPLE 1 Tissue Expression Analysis of FABP4, FABPS, and PPARγ in HumanPsoriatic Skin Lesions

Punch biopsies (4 mm in diameter) were obtained from disease-involvedskin of patients with psoriasis (n=10). Biopsies were further obtainedfrom involved skin of patients with chronic dermatitis (n=5). Normalskin was obtained from patients after surgical reduction of redundantskin (n=10). The tissues were fixed in formalin and embedded inparaffin. For histopathological examination with light microscopy, thesections were stained with hematoxylin and eosin (H&E) and observed by apathologist who confirmed the diagnosis. In addition, forimmunohistochemical analysis, the sections were stained with thefollowing antibodies: FABP4 (Rabbit polyclonal anti FABP4 antibody,PAB12276, Abnova), FABPS (Rabbit polyclonal anti FABPS antibody,SC-50379, Santa Crus) and PPARγ (mouse monoclonal anti-PPARγ antibody,E-8, Santa Crus), all diluted 1:50. The paraffin-embedded andcryopreserved tissues were processed according to a standard protocol.

FIG. 1C shows high expression levels of FABP4 as detected in psoriaticskin lesions compared to normal skin (FIG. 1A), both in the epidermisand the dermis. This finding was observed in all the psoriasis-involvedbiopsies that were tested (100 percent prevalence). To the best of theinventors' knowledge, this is the first time that the expression ofFABP4 in keratinocytes and dermal cells in psoriatic lesions has beenreported. Without wishing to be bound by theory, this finding maysuggest that FABP4 overexpression is related to the dysregulation ofkeratinocyte proliferation and differentiation in psoriasis. Inaddition, this observation supports a role of FABP4 in promotinginflammation by its action in dermal immune cells, which is anothersignificant feature of psoriasis.

Increased level of FABP4 in both keratinocytes and inflammatory cells inthe dermis were also observed in skin biopsies from patients withchronic dermatitis, as shown in FIG. 1E, although less than in psoriasis(FIG. 1C).

Moreover, a negative correlation between FABP4 and PPARy expression wasobserved in keratinocytes and immune cells: while in normal skin PPARywas detected throughout the epidermis and in few cells in the dermis, asshown in FIG. 1B, in psoriatic lesions the expression of PPARy wassignificantly reduced and was displayed only in the uppermost layers ofthe epidermis, and not detected in dermal cells (FIG. 1D). PPARyexpression was also reduced in dermatitis skin biopsies, as shown inFIG. 1F. FABPS was expressed only in keratinocytes from psoriatic skin,as previously reported ([16], not shown).

Psoriatic skin is characterized by a hyperproliferative epidermiscomprised from multiple layers of keratinocytes, resulting in increasedthickness of the skin. At higher magnification, FABP4 was demonstratedthroughout the hyperplastic epidermis in psoriatic lesions, whereinstaining was mostly cytoplasmic in the basal layers and nuclear in theupper layers, as shown in FIG. 2B. FABP4 expression in the dermis waspredominantly observed in macrophages, lymphocytes and endothelialcells, in a cytoplasmic pattern, as shown in FIG. 2C. PPARy expressionin psoriatic skin was markedly reduced in the epidermis, appeared onlyin the uppermost layers, and was undetected in the dermis compared as tonormal skin (see FIG. 3B versus 3A).

This suggests that the tendency to develop skin diseases having aninflammatory component may be detected by evaluating FABP, andparticularly FABP4, expression in dermis and epidermis skin samples. Italso suggests that FABP4 overexpression, and PPARy downregulation, arelinked to the pathogenesis of psoriasis.

EXAMPLE 2 Tissue Expression Analysis of FABP4 in Human Cutaneous T CellLymphoma (CTCL) Skin Lesions

CTCL is a group of neoplasms of skin-homing T cells. Mycosis fungoides(MF) represents the most common type of CTCL and accounts forapproximately 50% of all primary cutaneous lymphomas. Although malignantin nature, MF has a protracted clinical course with inflammatory,dermatitis-like presentation [8].

The expression of FABP4 among patients with MF was studied by performingan immunohistochemical analysis of lesional skin. Samples from 5patients with MF were tested and compared to normal human skin. Punchbiopsies (6 mm in diameter) were obtained from patients with MF, fromdisease-involved skin. Normal skin was obtained from patients aftersurgical reduction of redundant skin. Tissue sections were processed andstained with anti-FABP4 antibody as described in Example 1.

High expression levels of FABP4 were observed in MF lesions compared tonormal skin with all tested patients, as shown in FIGS. 4B and 4A,respectively. FABP4 expression was confined to the infiltrating Tlymphocytes in the epidermis and the dermis. A strong cytoplasmic stainwas observed in the dermal infiltrating cells, as well as in themalignant cells which penetrated the lower epidermis, which is thehallmark of MF.

Thus, due to the high expression of FABP4 protein in the malignant CTCLT-lymphocytes, detection of overexpression can be used to determine apatient's propensity towards development of CTCL or allow for earlydetection thereof.

Many dermatologic diseases (including psoriasis, dermatitis, and CTCL)are mediated by lymphocytes, predominantly T lymphocytes [17]. FABP4expression has been reported in a restricted repertoire of cell types,only in macrophages, adipocytes and endothelial cells. Inimmunohistochemistry analysis performed by the present inventor, FABP4was found to be expressed in inflammatory cells in the dermis, in bothmacrophages and lymphocytes. FABP4 expression in lymphocytes has notbeen reported so far, nor expression thereof in dermal cells in general,thus these findings are of high relevance in dermatological diseases.

EXAMPLE 3 Overexpressing FABP4 in Primary Murine Keratinocytes

The introduction of FABP4 into keratinocytes was suggested to create ahyperproliferative state with impaired differentiation as seen inpsoriasis. Differentiation was assessed by measuring the expression oftwo keratinocyte differentiation markers K1 and K5. K5 is a keratinwhose level does not change during keratinocyte differentiation andtherefore serves as a loading control, while K1 is induced during normalkeratinocyte differentiation.

For assessing the effect of FABP4 overexpression in mouse primarykeratinocytes, cells were infected with a lentiviral vector constructcontaining the FABP4 gene. The FABP4 lentiviral vector, namedFABP4-T2A-EGFP, was constructed with the FABP4 gene under the CMVpromotor, and contained the green fluorescence protein (GFP) gene as areporter of expression. The presence of a T2A residue at the N-terminusof the FABP4 protein precludes FABP4 detection by anti-FABP4 commercialantibodies, due to interference with their binding. Therefore, anti-GFPantibodies for validation of infection and FABP4 overexpression wereused instead.

For evaluating expression of K1 and K5 differentiation markers inprimary murine keratinocytes with or without FABP4 expression, primarykeratinocytes were prepared from neonatal mice as previously described[18], and infected with FABP4-T2A-EGFP vector. Two internal controlswere used: cells which were not infected with the lentivirus and cellsinfected with the EGFP vector lacking the FABP4 gene. The cells weregrown in differentiation media containing three different calciumconcentrations. Elevation of extracellular calcium from low (0.05 mM) tomedium (0.12 mM) or high (1 mM) concentration induced differentiation ofkeratinocytes.

Expression of keratinocyte differentiation markers K1 and K5 intransfected and non-transfected cells is demonstrated in FIG. 5:infection with the lentivirus expressing FABP4-T2A-EGFP reduced thelevel of K1 both in medium and high calcium conditions compared tonon-infected cells and GFP-infected cells. The level of K5 did notchange during keratinocyte differentiation in absence of infection, asexpected. The observation that FABP4 overexpression reduces K1 suggeststhat FABP4 interferes with the normal differentiation process ofkeratinocytes and promotes a hyperproliferative state, resemblingpsoriasis.

EXAMPLE 4 Inhibition of FABP4 in vivo in Imiquimod-Induced PsoriasisModel in Mice

As already explained, psoriasis is a chronic inflammatory skin disorder.It occurs when the immune system mistakes the skin cells as a pathogen,and sends out faulty signals that speed up the proliferation of skincells. Imiquimod (IMQ) is a potent immune activator that induces andexacerbates psoriasis when applied topically. Daily application of IMQon mouse back skin induces inflamed scaly skin lesions resembling plaquetype psoriasis [19, 20]. IMQ-induced psoriasis in mice has been longused as a model for human psoriasis.

The efficacy of oral administration of a FABP4-inhibitor to treatpsoriasis in the IMQ-induced model in mice was tested.

BMS309403(2-[[2′-(5-ethyl-3,4-diphenyl-1H-pyrazol-1-yl)[1,1′-biphenyl]-3-yl]oxy]-aceticacid, Formula I) was used as a FABP4-inhibitor:

The test was carried out using Balb/c mice (Envigo RMS (Israel) Ltd),average (±SD) body weight at study initiation was 18.2±0.81 g. Animalswere fed ad libitum a commercial rodent diet (Teklad Certified Global18% Protein Diet, Harlan cat# 2018SC). Animals had free access tosterilized and acidified drinking water (pH between 2.5 and 3.5).

The tests included six groups of 3-10 mice per group, as follows:

-   -   one naïve (no IMQ) group;    -   one Vehicle control group, the vehicle formulation being 10%        1-methyl-2-pyrrolidone and 5% Cremophor EL in water for        injection (WFI);    -   one treated group that received Cortisone acetate as a positive        control—one tablet (25 mg each, Rekah Pharm) was triturated with        a mortar. The powder was dissolved in 2.5 ml WFI yielding 10        mg/ml. The compound was administrated per-os at 12.5 mg/kg BW        (body weight) mouse of the compound once daily for six days        starting the first day of IMQ application (Day 1); and    -   three treated groups that received BMS (i.e. Test Item) at three        doses—5, 15 and 30 mg/kg. BMS powder (Cayman Chemical) was        dissolved in ethanol to create a 30 mg/ml solution. This stock        solution was diluted in the Vehicle to create three different        concentrations 0.5, 1.5 and 3 mg/ml. A new aqueous solution was        prepared in each day. BMS was administrated per-os once daily        for six days starting the first day of IMQ application (Day 1).

IMQ-induction: A daily topical dose of 62.5 mg of commercially availableIMQ cream (5%) (Aldara; 3M Pharmaceuticals) was applied to animals fromall the groups except for the naive group on the shaved back for sixconsecutive days, translated to a daily dose of 3.125 mg of the activecompound.

Experiment conditions: All animals except for the naive group receivedIMQ cream (Aldara™ 5% cream, #3 Pharmaceuticals) applied on the backskin daily for six consecutive days starting on Day 1 (˜60 mg/mouse).The Test Item, Vehicle and Cortisone acetate were administered per-osdaily for 6 days. During the study morbidity and mortality, body weight(BW), clinical signs, and Psoriasis Area and Severity Index (PASI)scoring were performed and representative pictures were taken. Theanimals were sacrificed on Day 9.

Scoring was performed by a trained observer in a “blinded” way, i.e.,being unaware of the treatment. To score the severity of inflammation ofthe back skin, the clinical PASI scoring was employed. Erythema,scaling, and thickening were scored independently on a scale from 0 to4: 0, none; 1, slight; 2, moderate; 3, marked; 4, very marked. Thecumulative score (erythema plus scaling plus thickening), PASI scoring,served as a measure of the severity of inflammation (scale 0-12).

Mortality & morbidity: During both experiments, no animal died or wasfound in morbid conditions. No abnormal clinical signs were observed inany of the animals during the study. No effect of the treatment on bodyweight was detected.

Scoring severity of skin inflammation: The average score of the severityof inflammation of the back skin, i.e. the PASI, is presented in FIG. 6.Each parameter, namely erythema severity, skin thickness, and scaling,was measured individually (FIGS. 7-9, respectively). According to thePASI scoring, all IMQ treated groups developed considerable skinreaction, as compared to the naïve group, notably on Days 5 and 7 of thestudy. On Day 7 significant amelioration of the skin inflammation by theBMS treatment was observed. The middle dose of BMS (15 mg/kg) seemed tohave the highest impact on the inflammation severity. This doseexhibited the best results on the tested parameters—erythema, skinthickness and scaling. Representative pictures taken at Day 7 are shownin FIGS. 10A-10F.

In summary, all IMQ treated groups developed considerable skin reaction,as compared to the naïve group, notably on Days 5 and 7 of the study. OnDay 7 significant amelioration of the effect was observed in the BMStreated groups for all test parameters, i.e. inflammation severity,erythema, skin thickness and scaling. The experiment was repeated treetimes with similar results.

1. A pharmaceutical composition comprising at least one FABP4-inhibitorfor use in treating or preventing a skin disease.
 2. The pharmaceuticalcomposition of claim 1, wherein said at least one FABP4-inhibitor isselected from a peptide, an antibody, an antibody fragment, a smallmolecule, a small interfering RNA (siRNA), a small hairpin RNA (shRNA),and mixtures thereof.
 3. The pharmaceutical composition of claim 2,wherein said at least one FABP4-inhibitor is selected from the groupconsisting of (i) a small molecule having a molecular weight of up toabout 1000 Da, or up to 500 Da, (ii) an antibody that specifically bindsto and inhibits the activity of FABP4, (iii) an siRNA comprising thenucleic acid sequences SEQ ID NO: 2-9. 4-6. (canceled)
 7. Thepharmaceutical composition of claim 1 wherein said FABP4-inhibitor isalso a FABP5-inhibitor.
 8. The pharmaceutical composition of claim 1wherein said skin disease is selected from the group consisting ofpsoriasis, dermatitis (atopic, seborrheic, contact), eczema,parapsoriasis, lichen planus, lichen plano-pilaris, pityriasislichenoides et varioliformis acuta, pityriasis lichenoides chronica,pityriasis rubra pilaris, pityriasis rosea, graft-versus-host disease,histiocytoses, drug-induced eruptions, autoimmune connective tissuediseases (e.g. lupus), rosacea, folliculitis, acne, warts, ichthyosis,vitiligo, scarring alopecia, CTCL, actinic keratosis, squamous cellcarcinoma, basal cell carcinoma, nevus, lichen simplex chronicus,xerosis, keratosis, keratoderma, pruritus, a burn, a scar, a callus, anda keloid.
 9. (canceled)
 10. The pharmaceutical composition of claim 1,wherein the skin disease is an inflammatory skin disease. 11-13.(canceled)
 14. The pharmaceutical composition of claim 1, adapted fordelivery of said at least one FABP-inhibitor topically, orally, byinhalation, nasally, transdermally, ocularly or parenterally into thecirculatory system of a subject. 15-20. (canceled)
 21. Thepharmaceutical composition of claim 1, further comprising a PPARyagonist or adapted for administration together with a PPARy agonist.22-24. (canceled)
 25. A method for treating or preventing a skin diseasein a subject, comprising administering to a subject in need thereof, atherapeutically effective amount of at least one FABP4-inhibitor or apharmaceutical composition comprising thereof.
 26. The method of claim25, further comprising administering a PPARy agonist to the subject. 27.The method of claim 26, wherein said PPARy agonist is administeredconcomitantly with said at least one FABP4-inhibitor.
 28. The method ofclaim 26, wherein said FABP-inhibitor and said PPARy agonist areadministered sequentially.
 29. The method of claim 28, wherein said atleast one FABP4-inhibitor is selected from a peptide, an antibody, asmall molecule, a small interfering RNA (siRNA), a small hairpin RNA(shRNA), and mixtures thereof.
 30. The method of claim 29, wherein saidat least one FABP4-inhibitor is selected from the group consisting of(i) a small molecule has a molecular weight of up to about 1000 Da, orup to 500 Da, (ii) an antibody that specifically binds to and inhibitsthe activity of FABP4, (iii) an siRNA comprising the nucleic acidsequences SEQ ID NO: 2-9. 31-33. (canceled)
 34. The method of claim 25,wherein said FABP4-inhibitor is also a FABP5 inhibitor
 35. The method ofclaim 25, wherein said skin disease is selected from the groupconsisting of psoriasis, dermatitis (atopic, seborrheic, contact),eczema, parapsoriasis, lichen planus, lichen plano-pilaris, pityriasislichenoides et varioliformis acuta, pityriasis lichenoides chronica,pityriasis rubra pilaris, pityriasis rosea, graft-versus-host disease,histiocytoses, drug-induced eruptions, autoimmune connective tissuediseases (e.g. lupus), rosacea, folliculitis, acne, warts, ichthyosis,vitiligo, scarring alopecia, CTCL, actinic keratosis, squamous cellcarcinoma, basal cell carcinoma, nevus, lichen simplex chronicus,xerosis, keratosis, keratoderma, pruritus, a burn, a scar, a callus, anda keloid.
 36. (canceled)
 37. The method of claim 25, wherein the skindisease is an inflammatory skin disease. 38-41. (canceled)
 42. A methodfor detecting the predisposition in a subject for developing a skindisease or condition, comprising: detecting expression of FABP4 in asample containing keratinocytes or inflammatory cells from the subject,wherein the presence of FABP4 in the sample above a pre-determinedbase-level indicates a predisposition for developing the skin disease orcondition.
 43. The method of claim 42, wherein the sample is a skinsample and said pre-determined base-level is a level of FABP4 in anormal skin sample.
 44. (canceled)
 45. The method of claim 42, whereindetecting comprises detecting the expression of a FABP4 nucleic acid.46. (canceled)
 47. The method of claim 42, wherein said skin disease isselected from the group consisting of psoriasis, dermatitis (atopic,seborrheic, contact), eczema, parapsoriasis, lichen planus, lichenplano-pilaris, pityriasis lichenoides et varioliformis acuta, pityriasislichenoides chronica, pityriasis rubra pilaris, pityriasis rosea,graft-versus-host disease, histiocytoses, drug-induced eruptions,autoimmune connective tissue diseases (e.g. lupus), rosacea,folliculitis, acne, warts, ichthyosis, vitiligo, scarring alopecia,CTCL, actinic keratosis, squamous cell carcinoma, basal cell carcinoma,nevus, lichen simplex chronicus, xerosis, keratosis, keratoderma,pruritus, a burn, a scar, a callus, and a keloid. 48-52. (canceled)